Comparison of functional limbal epithelial stem cell isolation methods

Abstract

Purpose: The corneal epithelium is renewed by limbal epithelial stem cells (LESC), whose dysfunction results in loss of vision and pain. The current treatment of choice is the transplantation of in vitro cultured LESC. However, the most optimal technique of LESC isolation from the limbal niche has not been well established yet. Our aim was to determine which isolation method renders the highest recovery of functional LESC from the human limbus.

Methods: We compared limbal primary cultures (LPC) obtained from explants and cell suspensions. Cell morphology was observed by phase contrast and transmission electron microscopy. LESC (K14, K15, ABCG2, and p63α), corneal epithelial cell (K3 and K12) and fibroblast (S100A4) markers were analyzed by immunofluorescence and real time RT-PCR. Markers of endothelial cells (PECAM), melanocytes (MART-1), dendritic cells (CD11c), and proliferation stage (Ki67) were analyzed by immunofluorescence. Colony forming efficiency (CFE) and the presence of holoclones, meroclones, and paraclones were also studied.

Results: LPC cells obtained from both methods had cuboidal morphology, a sparse cytoplasm, definited nucleoli, desmosomes, microvilli, and prominent intermediate filaments. The expression of LESC markers (K14, K15, ABCG2, p63α) was similar or higher in LPC established through cell suspensions, except the expression of p63α mRNA that was higher in LPC generated from explants. There were no significant differences in the expression of corneal epithelial markers (K3, K12). PECAM, MART-1, and CD11c proteins were not detected, while fibroblast-protein (S100A4) was mildly detected in all LPC. The percentage of positive cells for the proliferation marker (Ki67) was similar in LPC established from either technique, while the CFE was significantly higher in LPC from cell suspensions. Cells derived from confluent LPC produced by explants generated only paraclones (100%), while the percentage of paraclones derived from LPC established from cell suspensions was 90% and the remaining 10% were meroclones.

Conclusions: LPC established from cell suspensions have a cell population richer in functional LESC than LPC obtained from explants, suggesting than in a clinical situation in which it is possible to choose, the cell suspension method is probably the best option.